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Olfactory sensitivity to changes in environmental [Ca²⁺] in the freshwater teleost Carassius auratus: an olfactory role for the Ca²⁺-sensing receptor?

P. C. Hubbard^1,*, P. M. Ingleton², L. A. Bendell², E. N. Barata^1,3 and A. V. M. Canário¹

¹ Centro de Ciências do Mar, Universidade do Algarve, Campus de Gambelas, 8000-810 Faro, Portugal
² Division of Genomic Medicine, Academic Unit of Endocrinology, University of Sheffield, The Medical School, Beech Hill Road, Sheffield S10 2TN, UK
³ Departamento de Biologia, Universidade de Évora, Apartado 94, 7001 Évora Codex, Portugal

View larger version (22K): [in a new window]   Fig. 1. Olfactory responses of Carassius auratus to changes in environmental [Ca2+]. (A) Typical electroencephalogram (EEG) recordings from the olfactory bulb (lower traces) and their integrated activity (upper traces) in response to increases in [Ca2+] from nominally zero (Ca2+-free AFW) to 0.1, 1.0 and 10 mmol l-1 (horizontal bars). All three recordings were taken from the same fish. (B) Semi-logarithmic plot of mean values (normalised data, see Materials and Methods for details; N=6) showing the concentration dependence of this effect (r2=0.98), and a Hill plot fitted to the data giving an apparent EC50 of 0.6 mmol l-1 [Ca2+] and a Hill coefficient of 0.8. The environmental range of [Ca2+] normally encountered by Carassius auratus and the plasma free [Ca2+] are also shown.

View larger version (20K): [in a new window]   Fig. 2. Olfactory responses of Carassius auratus to changes in environmental [Na+], [Mg2+] and [K+]. (A) Typical electroencephalogram (EEG) recordings from the olfactory bulb (lower traces) and their integrated activity (upper traces) in response to 100 mmol l-1 Na+ (in Na+-free AFW), 100 mmol l-1 Mg2+ in AFW and 10 mmol l-1 K+ in AFW (horizontal bars). All three recordings were taken from the same fish. (B) Semi-logarithmic plot of pooled, normalised (to the response to 10-5 mol l-1 L-serine in AFW) data of integrated EEG amplitude (means ± S.E.M.; N=5-6) in response to changes in [Ca2+] (filled circles), [Mg2+] (filled triangles), [Na+] (open circles) and [K+] (open triangles). Note that only the responses to Na+ approach the amplitude of those evoked by Ca2+, and then only at high concentrations.

View larger version (19K): [in a new window]   Fig. 3. Effects of cross adaptation to Mg2+ on olfactory responses to Ca2+ in Carassius auratus. Two consecutive olfactory electroencephalogram (EEG) responses (lower traces) and their integrated activities (upper traces) to 10 mmol l-1 Ca2+ (black horizontal bars) in the absence (open horizontal bar) and presence (shaded horizontal bar) of 100 mmol Mg2+. Note that the response to calcium is bluntened, but not abolished, in the presence of excess Mg2+.

View larger version (25K): [in a new window]   Fig. 4. Effects of removal of external Ca2+ and Na+ on olfaction in Carassius auratus. (A) Typical electroencephalogram (EEG) responses from the olfactory bulb (lower traces) and their integrated activity (upper traces) to 10-5 mol l-1 L-serine (black horizontal bars) in AFW (open horizontal bar), to Ca2+-free and to Na+-free (shaded horizontal bars) AFW flowing over the olfactory epithelium. All three responses were recorded from the same fish. (B,C) Histograms showing pooled data (means + S.E.M.) of responses to 10-5 mol l-1 L-serine in Ca2+-free AFW (B) and Na+-free AFW (C) compared with their respective controls (10-5 mol l-1 L-serine in AFW). The number of observations is shown in parentheses. *P<0.05 (Student's t-test for paired data).

View larger version (120K): [in a new window]   Fig. 5. Ca2+-sensing receptors (Ca-SR) in goldfish olfactory epithelium. (A) Immunocytochemistry with rabbit antiserum to an epitope of the external domain of puffer fish Ca-SR. There is a relatively low concentration of receptors in the cells of the outermost layer of the epithelium (e) of a primary lamella with isolated, apparently migratory receptor-type cells (some indicated by open arows) staining more intensely, indicating a much higher receptor content. Magnification x350. (B) Negative control reaction in which the specific primary antibody was replaced by normal rabbit serum. No reaction was detected in any cells of the olfactory epithelium. Magnification x350. (C) In situ hybridisation of Ca-SR with a DIG-labelled oligonucleotide probe, showing abundant hybridisation in the basal layer of the olfactory epithelium (open arrows). Magnification x350. (D) A negative control in situ hybridisation in a similar area of epithelium to that in C; the specific oligoprobe was omitted from the reagents. There was no reaction in any olfactory tissue cells. Magnification x350.